Apoptotic compounds

ABSTRACT

The invention provides methods and compositions for enhancing apoptosis of pathogenic cells. The general method comprises the of contacting the cells with an effective amount of an AV peptoid, wherein the AV peptoid is a peptide comprising AX 1 , wherein X 1  is V, I or L, or a peptide mimetic thereof, which interacts with an Inhibitor of Apoptosis protein (IAP) as measured by IAP binding, procaspase-3 activation or promotion of apoptosis, wherein apoptosis of the pathogenic cells is enhanced. The subject compositions encompass pharmaceutical compositions comprising a therapeutically effective amount of a subject AV peptoid in dosage form and a pharmaceutically acceptable carrier, wherein the AV peptoid is a peptide comprising AX 1 , wherein X 1  is V, I or L, or a peptide mimetic thereof, which inhibits the activity of an Inhibitor of Apoptosis protein (IAP) as measured by IAP binding, procaspase-3 activation or promotion of apoptosis. The invention also provides assays for identifying agents which modulates the interaction of an AV peptoid with an IAP, active compounds identified in such screens and their use in the foregoing compositions and therapeutic methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of Ser. No. 10/641,539, filed Aug. 15,2003, which is a continuation of Ser. No. 09/645,075, filed Aug. 23,2000 now U.S. Pat. No. 6,608,026.

This invention was made with Government support under Grant No.GMRO1-57158, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

INTRODUCTION

1. Field of the Invention

The field of the invention is promoting cell death.

2. Background

Apoptosis plays a central role in the development and homeostasis of allmulti-cellular organisms1-4. Abnormal inhibition of apoptosis is ahallmark of cancer and autoimmune diseases, whereas excessive activationof cell death is implicated in neuro-degenerative disorders such asAlzheimer's disease^(5, 6). In fact, one mode of action ofchemotherapeutic drugs is via the activation of apoptosis; understandinghow the cell death program is engaged following an insult, and hence whyit fails to be engaged in certain settings, offers a novel approach toovercoming the clinical problem of drug resistance; see, e.g. Makin etal., Cell Tissue Res 2000 July;301(1):143–52 (“Apoptosis and cancerchemotherapy”).

The mechanism of apoptosis is conserved across species and executed witha cascade of sequential activation of initiator and effectorcaspases^(7, 8). Caspases, a family of cysteine proteases with aspartatesubstrate specificity, are produced in cells as catalytically inactivezymogens⁷. Once activated, the effector caspases are responsible forproteolytic cleavage of a broad spectrum of cellular targets thatultimately lead to cell death.

The Inhibitor of Apoptosis (IAP) family of proteins suppress apoptosisby preventing the activation of procaspases and inhibiting the enzymaticactivity of mature caspases^(9, 10). Several distinct mammalian IAPsincluding XIAP, c-IAP1, c-IAP2, and survivin, have been identified, andthey all exhibit anti-apoptotic activity in cell culture^(9, 10). InDrosophila, the anti-apoptotic activity of IAPs is removed by Reaper,Grim, and Hid, all of which appear to act upstream of IAPs andphysically interact with IAPs to relieve their inhibitory effect oncaspase activation^(11, 12). IAPs are known to be overexpressed in humancancers²⁶⁻³³.

One major caspase activation cascade is triggered by the release ofcytochrome c from the intermembrane space of mitochondria¹³⁻¹⁹.Concurrent with cytochrome c release, another regulator of apoptosis,Smac²⁰ (Second mitochondria-derived activator of caspases) or DIABLO²¹,is also released from the mitochondria into the cytosol. Smac eliminatesthe inhibitory effect of multiple IAPs and interacts with all IAPs thathave been examined, including XIAP, c-IAP1, c-IAP2, andsurvivin^(20, 21).

Smac is synthesized as a precursor molecule of 239 amino acids; theN-terminal 55 residues serve as the mitochondria targeting sequence thatis removed after import²⁰. The mature form of Smac contains 184 aminoacids and behaves as an oligomer in solution²⁰. We recently found thatthe 2.2 Å resolution crystal structure of the mature form of Smacreveals an arch-shaped homo-dimer with rich surface features (the atomiccoordinates are being deposited with the Protein Data Bank with theaccession number 1FEW). The homo-dimeric interface is dominated byhydrophobic residues through van der Waals interactions. Mutations ofkey residues at the interface disrupted dimer formation andsignificantly weakened the ability of Smac to induce the activation ofprocaspase-3 and to promote the enzymatic activity of mature caspase-3.In addition, similar to the Drosophila proteins Reaper, Grim, and Hid,the N-terminal amino acids of Smac/DIABLO were indispensable for itsfunction; in fact, mutation of the very first amino acid rendered theresulting protein completely inactive. The sequence homology amongReaper, Grim, and Hid is restricted to their N-terminal 14 amino acids;deletion of these residues led to loss of interaction with IAPs⁹ and afusion protein comprising the N-terminal 37-residue peptide of Hidinduced apoptosis in insect cells¹¹. Here we further disclose smallpeptides, and peptide mimetics that are sufficient to bind IAP, promoteactivation of procaspase-3 and/or promote apoptosis.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for enhancing apoptosisof pathogenic cells. The general method comprises the of contacting thecells with an effective amount of an AV peptoid, wherein the AV peptoidis a peptide comprising AX₁, wherein X₁ is V, I or L, or a peptidemimetic thereof, which interacts with an Inhibitor of Apoptosis protein(IAP) as measured by IAP binding, procaspase-3 activation or promotionof apoptosis, wherein apoptosis of the pathogenic cells is enhanced.

In some embodiments, the cells are in situ in an individual and thecontacting step is effected by administering to the individual apharmaceutical composition comprising a therapeutically effective amountof the. AV peptoid, wherein the individual may be subject to concurrentor antecedent radiation or chemotherapy for treatment of aneoproliferative pathology. In other embodiments, the pathogenic cellsare of a tumor selected from the group consisting of breast cancer,prostate cancer, lung cancer, pancreatic cancer, gastric cancer, coloncancer, ovarian cancer, renal cancer, hepatoma, melanoma, lymphoma, andsarcoma. In yet other embodiments, the AV peptoid is a peptidecomprising AX₁X₂, wherein X₁ is V, I or L and X₂ is P or A;particularly, comprising AX₁X₂, wherein X₁ is V and X₂ is P.

The subject compositions encompass pharmaceutical compositionscomprising a therapeutically effective amount of an AV peptoid in dosageform and a pharmaceutically acceptable carrier, wherein the AV peptoidis a peptide comprising AX₁, wherein X₁ is V, I or L, or a peptidemimetic thereof, which inhibits the activity of an Inhibitor ofApoptosis protein (IAP) as measured by IAP binding, procaspase-3activation or promotion of apoptosis.

In some embodiments, such compositions further comprise an additionaltherapeutic agent, such as an anti-neoproliferative chemotherapeuticagent, other than the AV peptoid. In other embodiments of suchcompositions, the AV peptoid is a peptide comprising AX₁X₂, wherein X₁is V, I or L and X₂ is P or A; particularly, comprising AX₁X₂, whereinX₁ is V and X₂ is P.

The invention also provides assays for identifying agents whichmodulates the interaction of an AV peptoid with an IAP, active compoundsidentified in such screens and their use in the foregoing compositionsand therapeutic methods. The general assay comprises the steps ofincubating a mixture comprising a subject AV peptoid, a secondbaculoviral IAP repeat domain (BIR2) of XIAP, and a candidate agent;under conditions whereby, but for the presence of said agent, thepeptoid specifically interacts with the BIR2 at a reference affinity;detecting a specific interaction of the peptoid with the BIR2 todetermine an agent-biased affinity, wherein a difference between theagent-biased affinity and the reference affinity indicates that theagent modulates the interaction of the peptoid to the BIR2 of the XIAP.

In some embodiments of the screen, the detecting step comprisesmeasuring in vitro binding of the peptoid to the BIR2 by pull-downassay, fluorescent polarization assay or solid-phase binding assay. Inother embodiments, the mixture further comprises procaspase-3 and acaspase-3 substrate and the detecting step comprises measuring theinteraction inferentially by detecting a reaction product of thecaspase-3 substrate and caspase-3 generated by activation of theprocaspase-3. In yet other embodiments, the incubating step comprisesincubating a cell comprising the mixture and the detecting stepcomprises measuring the interaction inferentially by detecting apoptosisof the cell, particularly wherein the cell is in situ in an animal host.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or and polynucleotide sequences are understood toencompass opposite strands as well as alternative backbones describedherein.

An AV peptoid is a peptide comprising AX₁, wherein X₁ is V, I or L, or apeptide mimetic thereof, which interacts with an Inhibitor of Apoptosisprotein (IAP) as measured by IAP binding, procaspase-3 activation orpromotion of apoptosis as described in the exemplified activity assaysbelow. In a more particular embodiment, the peptide comprises AX₁X₂,wherein X₁ is V, I or L preferably V and X₂ is P or A, preferably P. Thesubject AV peptoids are fewer than 20 residues (monomers), preferablyfewer than 10, more preferably fewer than 5 and preferably 2 or 3 inlength, with a molecular weight of less than about 1,000, preferablyless than about 500.

AV peptoids include peptide mimetics of the subject peptides. A peptidemimetic is a non-naturally occurring analog of a peptide which, becauseof protective groups at one or both ends of the mimetic, or replacementof one or more peptide bonds with non-peptide bonds, is less susceptibleto proteolytic cleavage than the peptide itself. For instance, one ormore peptide bonds can be replaced with an alternative type of covalentbond (e.g., a carbon--carbon bond or an acyl bond). Peptide mimetics canalso incorporate amino-terminal or carboxyl terminal blocking groupssuch as t-butyloxycarbonyl, acetyl, alkyl, succinyl, methoxysuccinyl,suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl,fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl,and 2,4,-dinitrophenyl, thereby rendering the mimetic less susceptibleto proteolysis. Non-peptide bonds and carboxyl- or amino-terminalblocking groups can be used singly or in combination to render themimetic less susceptible to proteolysis than the corresponding peptide.Additionally, substitution of D-amino acids for the normalL-stereoisomer can be effected, e.g. to increase the half-life of themolecule. Accordingly, the peptide mimetics include peptides having oneor more of the following modifications:

peptides wherein one or more of the peptidyl [—C(O)NR—] linkages (bonds)have been replaced by a non-peptidyl linkage such as a —CH₂-carbamatelinkage [—CH₂—OC(O)NR—]; a phosphonate linkage; a —CH₂-sulfonamide[—CH₂—S(O)₂NR—] linkage; a urea [—NHC(O)NH—] linkage; a —CH₂-secondaryamine linkage; or an alkylated peptidyl linkage [—C(O)NR⁶— where R⁶ islower alkyl];

peptides wherein the N-terminus is derivatized to a —NRR¹ group; to a—NRC(O)R group; to a —NRC(O)OR group; to a —NRS(O)₂R group; to a—NHC(O)NHR group, where R and R¹ are hydrogen or lower alkyl with theproviso that R and R¹ are not both hydrogen; to a succinimide group; toa benzyloxycarbonyl-NH—(CBZ-NH—) group; or to a benzyloxycarbonyl-NE-group having from 1 to 3 substituents on the phenyl ring selected fromthe group consisting of lower alkyl, lower alkoxy, chloro, and bromo; or

peptides wherein the C terminus is derivatized to —C(O)R² where R² isselected from the group consisting of lower alkoxy, and —NR³R⁴ where R³and R⁴ are independently selected from the group consisting of hydrogenand lower alkyl.

Preferred mimetics have from zero to all of the —C(O)NH— linkages of thepeptide replaced by a linkage selected from the group consisting of a—CR₂OC(O)NR-linkage; a phosphonate linkage; a —CH₂S(O)₂NR— linkage; a—CH₂NR— linkage; and a —C(O)NR⁶— linkage, and a —NHC(O)NH— linkage whereR is hydrogen or lower alkyl and R⁶ is lower alkyl,

and wherein the N-terminus of the mimetic is selected from the groupconsisting of a —NRR¹ group; a —NRC(O)R group; a —NRC(O)OR group; a—NRS(O)₂R group; a —NHC(O)NHR group; a succinimide group; abenzyloxycarbonyl-NH— group; and a benzyloxycarbonyl-NH— group havingfrom 1 to 3 substituents on the phenyl ring selected from the groupconsisting of lower alkyl, lower alkoxy, chloro, and bromo, where R andR¹ are independently selected from the group consisting of hydrogen andlower alkyl,

and still further wherein the C-terminus of the mimetic has the formula—C(O)R² where R² is selected from the group consisting of hydroxy, loweralkoxy, and —NR³R⁴ where R³ and R⁴ are independently selected from thegroup consisting of hydrogen and lower alkyl and where the nitrogen atomof the —NR³R⁴ group can optionally be the amine group of the N-terminusof the peptide so as to form a cyclic peptide,

and physiologically acceptable salts thereof.

An important aspect of the invention is drawn to peptoids comprisingN-substituted glycine analogs which resemble naturally-occurring aminoacids (i.e., Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr) and comprising the generalformula I: X_(n)NRCH₂COOX_(c), wherein the radicals X_(n) and X_(c) areeither chains of conventional amino acids, chains of one or moreN-substituted glycine analogs, or chains in which conventional aminoacids and N-substituted glycine analogs are interspersed.

Preferred N-substituted glycine analogs are those in which R is ethyl,prop-1-yl, prop-2-yl, 1-methylprop-1-yl, 2-methylprop-1-yl, benzyl,4-hydroxybenzyl, 2-hydroxyethyl, mercaptoethyl, 2-aminoethyl,3-aminopropyl, 4-aminobutyl, 2-methylthioeth-1-yl, carboxymethyl,2-carboxyethyl, carbamylmethyl, 2-carbamylethyl, 3-guanidinoprop-1-yl,imidazolylmethyl, or indol-3-yl-ethyl, particularly where R is2-methylpropyl, benzyl, 2-hydroxyethyl, 2-aminoethyl, or carboxymethyl.The resemblance between amino acid and substitute need not be exact. Forexample, one may replace lysine with compounds of formula I in which Ris aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,5-aminopentyl, or 6-aminohexyl. Serine may be replaced withhydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and thelike. In general, a conventional amino acid may be replaced with anN-substituted glycine analog having a sidechain of similar character,e.g, hydrophobic, hydrophilic, polar, nonpolar, aromatic, etc.

Monomer refers to a molecule which may be linked to other monomers toform a peptoid. Monomers include amino acid substitutes, which mayinclude N- and/or C-terminal modifications to facilitate linking, forexample, leaving or activating groups.

N-substituted glycine analog refers to compounds of the formulaRNH—CH₂—COOH, where R is as defined above. The salts and esters of thesecompounds, as well as compounds of the formula bearing standardprotecting groups (e.g., Fmoc, t-Boc, and the like) are also consideredwithin the definition of “monomer” and “N-substituted glycine analog”unless otherwise specified.

A peptoid of the invention corresponds to a natural peptide if itelicits a biological activity related to the biological activity of thenatural protein. The elicited activity may be the same as, greater thanor less than that of the natural protein, i.e., provide enhanced and/orblocking effects. In general, such a peptoid will have an essentiallycorresponding monomer sequence, where a natural amino acid is replacedby an N-substituted glycine derivative, if the N-substituted glycinederivative resembles the original amino acid in hydrophilicity,hydrophobicity, polarity, etc. Thus, the following pairs of peptoidswould be considered corresponding in monomer sequence:

Ia: Ala-Val-Ile Ib: Ala-Val*-Ile* IIa: Ala-Ile-Pro-Gly-Phe-Ser-Pro-Phe(SEQ ID NO: 1) IIb: Ala-Ile-Pro-Gly-Phe*-Ser*-Pro-Phe* (SEQ ID NO: 2)IIIa: Ala-Leu-Phe-Met-Thr (SEQ ID NO: 3) IIIb: Ala-Leu-Phe*-Met-Ser*(SEQ ID NO: 4)In these examples, “Val*” refers to N-(prop-2-yl)glycine, “Phe*” refersto N-benzylglycine, “Ser*” refers to N-(2-hydroxyethyl)glycine, “Leu*”refers. to N-(2-methylprop-1-yl)glycine, and “Ile*” refers toN-(1-methylprop-1-yl)glycine.

The correspondence need not be exact: for example,N-(2-hydroxyethyl)glycine may substitute for Ser, Thr, Cys, and Met;N-(2-methylprop-1-yl)glycine may substitute for Val, Leu, and Ile. Notein IIIa and IIIb above that Ser* is used to substitute for Thr and Ser,despite the structural differences: the sidechain in Ser* is onemethylene group longer than that of Ser, and differs from Thr in thesite of hydroxy-substitution. In general, one may use anN-hydroxyalkyl-substituted glycine to substitute for any polar aminoacid, an N-benzyl- or N-aralkyl-substituted glycine to replace anyaromatic amino acid (e.g., Phe, Trp, etc.), an N-alkyl-substitutedglycine such as N-butylglycine to replace any nonpolar amino acid (e.g.,Leu, Val, Ile, etc.), and an N-(aminoalkyl)glycine derivative to replaceany basic polar amino acid (e.g., Lys and Arg).

The peptoids of the invention can be produced using amino acids as themonomer units or amino acid substitutes. Examples of differentmodifications in amino acids which can be carried out in order to obtainthe amino acid substitutes used in the invention are put forth below inTable 1.

TABLE 1 Peptoid Modification Chemistry Type of Iso- Enzyme H- ChiralModification steric Resistance Bonding Monomer I Peptoids IIN-alkylation + +++ + Yes III α-Ester +++ + +++ Yes IV Thioamide +++ ++ +Yes V N-hydroxylation + +++ + Yes VI β-Ester + ++ ++ Yes VIISulfonamide + ++ ++ No VIII Sulfonamide-N + ++ ++ No IX Urea + ++ ++ NoX Urethane + ++ ++ No Items II, III, IV and IX are taken from Spatola,A., “Peptide Backbone Modifications: . . .” in Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins (1983) 7: 267, B.Weinstein ed.. The + refers to the extent to which replacement ischaracterized by the given property: + = minimal, ++ = partial, +++ =substantial.

As can be seen from the table, modifications can significantly alter theproperties of the molecules, particularly with respect to enzymatichydrolysis. From a synthetic-standpoint, chiral starting materials canbe problematic. Even if they are easily synthesized, the fidelity of thesubsequent coupling reactions needs to be addressed. Each substituteamino acid structure is discussed briefly below and can be compared tothe amino acid structure in a peptide.

I. Peptide. The individual monomer units or substitute peptides such asthose described below can be combined together in any manner. However,it is most preferable to combine the monomer units using methodologysuch as disclosed in WO89/10931 in order to obtain large libraries ofdifferent peptoids, which libraries can then be screened to find one ormore peptoids which has a particular characteristic such as a highaffinity for a particular receptor site. Although the substitute aminoacids put forth below are examples of preferred substitute amino acidswhich can be used in connection with producing peptoids of theinvention, it should be noted that any monomer unit can be used whichwould allow for sequence specific synthesis of pools of diversemolecules. Any such monomer unit would be suitable for use in connectionwith the present invention in that such units would make it possible tosearch and screen for particular conformational shapes which haveaffinity for particular receptor sites. The use of nonpeptide polymersis believed to have particular advantages over conventional peptides inthat such peptoids would occupy different conformational configurationsin space and should provide resistance to the action of proteases, whichfeature would be particularly important to designing conjugates whereinthe peptoid portion would have a desirably long half-life. Further,substitute amino acids could be designed so as to provide for moleculeswhich are generally easier to synthesize than conventional peptidesmight be.

II. N-alkylated glycines. The main advantages of this system are theease of synthesis of the properly protected achiral monomers and thevast literature of peptides concerning the synthesis andcharacterization of the closely related peptoid polymers. The maindisadvantage is the decrease in properties dependent on the availabilityof amide protons for hydrogen bonding, such as solubility in aqueoussystems, conformational rigidity, secondary structure, etc. It ispointed out that N-alkylated glycines are a preferred class ofN-substituted glycines which can be used in connection with the presentinvention. Thus other chemically compatible groups other than R=alkylmay be used. Further, the substitutions may be made on the nineteenother natural amino acids.

III. α-Esters. Polyesters are one of the closest relatives to the normalpeptide bonds. The advantage is the close similarity, however, this canalso be a drawback since proteolytic enzymes are known to recognizeesters or even prefer esters as their substrates. α.-Polyesters areprepared from chiral .alpha.-hydroxy acids in which there has beenconsiderable synthetic interest (Chan, P. C., et al., Tetrahedron Lett(1990) 31:1985). In a stepwise fashion, polymers can be assembled muchas polyamides are prepared.

IV. Thioamides. The thioamide is also rather similar to the normalpeptide. According to Clausen, K., et al., J Chem Soc Perkin Trans(1984) 1:785, until 1984 there had been only limited reports of thethioamide replacement for a peptide bond which they attribute to thedifficulty in synthesis. He describes the synthesis and use of aprotected thioamide precursor using Lawessons's reagent. Also, a recentreport (Tetrahedron Lett (1990) 31:23) describes the conversion of apeptide bond to a thioamide using the same reagent.

V. N-hydroxy amino acids. The advantages are the decreased sensitivityto enzyme hydrolysis and H-bonding ability due to the added hydroxylgroup. Kolasa et al. has described the synthesis of N-hydroxypeptides(Kolasa, T., et al, Tetrahedron (1977) 33:3285).

VI. β-Ester. This is an example of a homologue of the .alpha.-ester.Presumably the different spacing will confer some special propertiessuch as increased resistance to enzyme hydrolysis or novelconformational flexibility. The appropriate starting materials arereadily synthesized (Elliott, J., et al., Tetrahedron Lett (1985)26:2535, and Tetrahedron Lett (1974) 15:1333.

VII. and VIII. Sulfonamides. The two sulfonamides differ by thepositioning of the R group. According to Frankel and Moses (Frankel, M.,et al., Tetrahedron (1960) 9:289), the peptide analog, i.e., the 1,4substituted polymer is not stable under their condensation conditions.Compounds of the type VII are readily obtained from chiral β-aminoalcohols (Kokotos, G., Synthesis (1990) 299) while those of the typeVIII are achiral and easily synthesized.

IX. Ureas. Ureas are also conveniently synthesized from carboxylic acidsand amines using the reagent diphenylphosphoryl azide, DPPA (Shiori, T.,et al., J Am Chem Soc (1972) 94:6203, and Bartlett, P., et al.,Synthesis (1989) 542). Previously prepared peptoids with a single ureareplacement had properties similar to the starting peptide (seereference 1, p. 231). Additionally, since there is still an amide protonavailable for H-bonding, the solubility properties may be better thanfor N-alkylated glycines.

X. Urethanes. The structure of a urethane is slightly different thanthat of a urea and would presumably have altered properties. Aqueoussolubility may be somewhat reduced since the amide proton is removed.The polymers may be prepared via simple chemistry.

There are numerous other polymer systems which could be employed for thepurpose of searching conformational space. Most notable are thephosphorous derived polymers with phosphonamides as one example(Yamauchi, K., et al., Bull Chem Soc Japan (1972) 45:2528). Polyamines(Tetrahedron Lett (1990) 31:23, and Kaltenbronn, J. S., et al., inProceedings of the Eleventh American Peptide Symposium (1989) 969, J.Rivier, ed.), polyalkanes, polyketones (Almquist, G., et al., J Med Chem(1984) 27:115, polythioethers, polysulfoxides (Spatola, A., et al.,Biopolymers (1986) 25:S229) and polyethers may be less suitable for ourpurposes due to either difficulty in synthesis or predictably poorproperties (e.g., polyamines would carry a positive charge at everyjunction and require double amine protection during synthesis). Insummary, several alternatives to N-alkylated glycine polymers of whichlibraries could be constructed have been described.

The foregoing examples of mimetics are nonlimiting. Peptide mimeticchemistry is a well-established art wherein skilled practitioners canreadily generate a wide variety of mimics using conventional chemistry(see, e.g. Liao et al. (1998) J. Med. Chem 41, 4767–4776; Andrade-Gordonet al. (1999) PNAS USA 96, 12257–12262; Boatman et al. (1999) J. Med.Chem. 42, 1367–1375; Kasher et al. (1999) J. Mol. Biol 292,421–429; U.S.Pat. No. 5,981,467; etc.) and these other strategies are applicablehere, so long as the resultant mimetics are screened for anddemonstrated to provide the requisite IAP inhibitory activity as assayedbelow.

Synthetic methods for producing the subject peptoids are well-known inthe art. Some general means for the production of peptides, analogs orderivatives are outlined in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, A Survey of Recent Developments, Weinstein, B.ed., Marcell Dekker, Inc., publ. New York (1983). A wide variety ofwell-established techniques are available for synthesizing peptidemimetics, see, e.g. submonomer method of R. Zuckermann et al., J. Am.Chem. Soc. (1992) 0114:10646–7. Synthesis by solid phase techniques ofheterocyclic organic compounds in which N-substituted glycine monomerunits forms a backbone is described in U.S. Pat. No. 5,958,792, whereincombinatorial libraries of mixtures of such heterocyclic organiccompounds can then be assayed for the ability to inhibit IAP asdescribed below. Highly substituted cyclic structures can be synthesizedon a solid support by combining the submonomer method with powerfulsolution phase chemistry. Cyclic compounds containing one, two, three ormore fused rings are formed by the submonomer method by firstsynthesizing a linear backbone followed by subsequent intramolecular orintermolecular cyclization, also as described in U.S. Pat. No.5,958,792. General preparative protocols for exemplary peptoid classesare as follows:

Preparation of α-Polyesters Using Chiral α-Hydroxy Acids As BuildingBlocks. The α-polyester structures can be prepared by using chemicalsynthesis technology known to those skilled in the art. For details ofthe reaction, see Brewster, P., et al., Nature, (1990) 166:179. Analternative method for producing similar structures is disclosed inChan, P. C., and Chong, J. M., Tetrahedron Lett. (1990)1985. Further,various publications cited within the Chan et al; publication describetechniques for synthesizing chiral α-hydroxy acids.

Preparation of Polythioamides Using Chiral .alpha.-Amino Acids AsBuilding Blocks. Polythioamide structures can be synthesized usingtechniques such as those described in Clausen, K., et al., J. Chem. Soc.Perkin Trans. I (1984) 785, and Tetrahedron Lett. (1990) 31:23

Preparation of Polyhydroxymates Using Chiral .alpha.-Amino Acids AsBuilding Blocks. Polyhydroxymates can be synthesized using techniques asdisclosed in Kolasa, T., and Chimiak, A., Tetrahedron (1977) 33:3285.References cited within Kolasa disclose and describe chemical techniquesfor synthesizing N-hydroxy amino acids which can be used in peptoidsynthesis.

Preparation of B-Polyesters Using Chiral β-Hydroxy Acids As BuildingBlocks. β-polyesters can be synthesized using a synthesis protocol asdescribed in Elliott, J. D., et al., Tetrahedron Lett. (1985) 26:2535,and Tetrahedron Lett. (1974) 15:1333.

Preparation of Polysulfonamides Using Chiral β-Amino Sulfonic Acids AsBuilding Blocks. Polysulfonamides can be synthesized using the reactionscheme shown in U.S. Pat. No. 6,075,121. The chiral β-amino acids havebeen described within Kokotos, G., Synthesis (1990) 299.

Preparation of N-alkylated Polysulfonamides Using Achiral β-AminoSulfonic Acids As Building Blocks. Similarly, these polysulfonamides canbe synthesized using the reaction scheme shown in U.S. Pat. No.6,075,121.

Preparation of Polyureas Using Achiral β-amino Acids As Building Blocks.Polyureas can be synthesized using techniques such as those described inShiori, T., et al., J. Am. Chem. Soc. (1972) 94:6302, and Scholtz, J.,and Bartlett, P., Synthesis (1989) 542.

Preparation of Polyurethanes Using Achiral β-Amino Alcohols As BuildingBlocks. Polyurethanes can be synthesized using the reaction scheme shownin U.S. Pat. No. 6,075,121. Individual N-substituted glycine analogs areknown in the art, and may be prepared by known methods. See, forexample, Sempuku et al., JP 58/150,562 (Chem Abs (1984) 100:68019b);Richard et al., U.S. Pat. No. 4,684,483; and Pulwer et al., EPO 187,130.

Several N-substituted glycine derivatives are available from commercialsources. For example, N-benzylglycine is available from Aldrich ChemicalCo. (Milwaukee, Wis.) as the ethyl ester. The ester is hydrolyzed inKOH/MeOH, then protonated in HCl to yield N-benzylglycine. This may thenbe protected with Fmoc (fluorenylmethoxycarbonyl) by treatment withFmoc-Cl in aqueous dioxane at high pH (about 10).

Other N-substituted glycine analogs are synthesized by simple chemicalprocedures. N-isobutylglycine may be prepared by reacting excess2-methylpropylamine with a haloacetic acid.

N-(2-aminoethyl)glycine may be prepared by reacting excess1,2-diaminoethane with a haloacetic acid and purifying on Dowex-1® (OHform), eluting with acetic acid. The unprotected amine is protected witht-butoxycarbonyl (t-Boc) using conventional techniques at pH 11.2,followed by protection of the secondary amine with Fmoc.

N-(2-hydroxyethyl)glycine may be prepared by reacting excess2-aminoethanol with haloacetic acid and purifying on Dowex-1® (OH form),eluting with acetic acid. The amine nitrogen is then protected withFmoc. Next, the acid group is esterified with methanol under acidicconditions. The methyl ester is then treated with isobutylene to formthe t-butyl ether. Then, the methyl ester is hydrolyzed using porcineliver esterase in phosphate buffer at pH 8.0, to provide a protectedN-substituted glycine analog in a form suitable for peptoid synthesis.As an alternative to the above, the Fmoc-hydroxyethylglycine is treatedwith t-butyldiphenylsilylchloride in DMF and imidazole to give asilyl-protected alcohol.

N-(carboxymethyl)glycine may be prepared by reacting glycine t-butylester with 2-haloacetate in aqueous solution. The product may beprotected directly by addition of Fmoc. As an alternative, theN-(carboxymethyl) glycine may be prepared by mixing glycine t-butylester, glyoxylic acid and palladium on charcoal under an atmosphere ofhydrogen in water at pH 6. The compound is then treated with FMOC in theusual manner.

Once the monomers have been synthesized, they may be coupled with othermonomers and/or conventional amino acids to form analogs using standardpeptide chemistry. For example, an Fmoc-protected monomer (N-substitutedglycine or conventional amino acid) may be immobilized on a suitableresin (e.g., HMP) by reaction withbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) or a carbodiimide (for example, dicyclohexylcarbodiimide) underbasic conditions (e.g., pH 9) in a suitable solvent. The Fmoc protectinggroup is removed by treatment with piperidine. Each additional monomeris then attached sequentially using BOP or a carbodiimide, until theentire sequence has been constructed. The completed chain is thendetached from the resin and the sidechain deprotected by treating withtrifluoroacetic acid (TFA).

Alternatively, one may connect N-substituted glycine analogs to the endsof peptoids produced by other methods, for example, by recombinantexpression or isolation from natural sources. Further, N-substitutedglycine analogs may be inserted within the sequence of such peptoids bycleaving the peptoid at the desired position, attaching an N-substitutedglycine analog, and reattaching the remainder of the molecule or achemically-synthesized replacement.

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions. In such compositions, the peptoid is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, thepeptoids may be advantageously used in conjunction with otherchemotherapuetic agents such as diethylstilbestrol or DES,5-fluorouracil, methotrexate, interferon-alpha, aspariginase, tamoxifen,flutamide, etc, and chemotherapeutic agents described in the MerckManuel, 16th edition 1992, Merck Research Laboratories, Rahway, N.J.;Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^(th)Ed., 1996, McGraw-Hill, esp. Chabner et al., Antineoplastic Agents atpp. 1233, etc. or otherwise known in the art. Hence the agents andpeptoids may be administered separately, jointly, or combined in asingle dosage unit. In a particular embodiment, the combination therapyis effected by a conjugate of the peptoid bound covalently to theanti-neoproliferative chemotherapeutic or other pharmaceutically activeagent. Any suitable conjugation chemistry may be used, such asderivatizing the N-terminus of the peptoids and conjugating the drugthrough an amid linkage.

The amount administered depends on the AV peptoid formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 0.1 mg to 1000 mg, preferably from about 1 mg to 300mg, more preferably 10 mg to 200 mg, according to the particularapplication. The actual dosage employed may be varied depending upon therequirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The following are examples (Examples 1–4) of capsule formulations forthe peptoids of Table 2.

TABLE 2 Capsule Formulations Formula Formula Formula Formula 1 mg/ 2 mg/3 mg/ 4 mg/ Capsule Formulation capsule capsule capsule capsule SolidSolution 100 400 400 200 Silicon Dioxide 0.625 2.5 3.75 1.875 MagnesiumStearate NF2 0.125 0.5 0.125 0.625 Croscarmellose 11.000 44.0 40.0 20.0Sodium NF Pluronic F68 NF 6.250 25.0 50.0 25.0 Silicon Dioxide NF 0.6252.5 3.75 1.875 Magnesium Stearate NF 0.125 0.5 1.25 0.625 Total 118.750475.00 475.00 475.00 Capsule Size No. 4 No. 0 No. 0 No. 2Preparation of Solid Solution

Crystalline peptoid (80 g/batch) and the povidone (NF K29/32 at 160g/batch) are dissolved in methylene chloride (5000 mL). The solution isdried using a suitable solvent spray dryer and the residue reduced tofine particles by grinding. The powder is then passed through a 30 meshscreen and confirmed to be amorphous by x-ray analysis.

The solid solution, silicon dioxide and magnesium stearate are mixed ina suitable mixer for 10 minutes. The mixture is compacted using asuitable roller compactor and milled using a suitable mill fitted with30 mesh screen. Croscarmellose sodium, Pluronic F68 and silicon dioxideare added to the milled mixture and mixed further for 10 minutes. Apremix is made with magnesium stearate and equal portions of themixture. The premix is added to the remainder of the mixture, mixed for5 minutes and the mixture encapsulated in hard shell gelatin capsuleshells.

AV peptoids can be administered by a variety of methods including, butnot limited to, parenteral, topical, oral, or local administration, suchas by aerosol or transdermally, for prophylactic and/or therapeutictreatment. The chemotherapeutic agent and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

The particular choice of peptoid, chemotherapeutic agent and/orradiation depends upon the diagnosis of the attending physicians andtheir judgement of the condition of the patient and the appropriatetreatment protocol. The peptoid, chemotherapeutic agent and/or radiationmay be administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,in any order, depending upon the nature of the proliferative disease,the condition of the patient, and the actual choice of chemotherapeuticagent and/or radiation to be administered in conjunction (i.e., within asingle treatment protocol) with the peptoid. Similarly, the peptoid andthe chemotherapeutic agent do not have to be administered in the samepharmaceutical composition, and may, because of different physical andchemical characteristics, be administered by different routes.

In one embodiment of the present invention, the method of the inventionincludes systemic or local administration of an AV peptoid. Wheresystemic administration is desired, the peptoid may be administered, forexample, by intravenous injection or orally. One embodiment of theinvention provides local administration of the peptoid, for example, atthe tumor site. With local administration of the peptoid, the preferredmode of administration is by local injection. However, localadministration may also be by catheter, or by local deposition, forexample by intra- or peritumoral administration of products sold underthe trademark Depofoam®, slow release pump/drug delivery service,implantable or topical gel or polymer, depending on the nature andlocation of the tumor. Administration of the therapeutics of theinvention can also be effectd by gene therapy protocol.

The therapeutics of the invention can be administered in atherapeutically effective dosage and amount, in the process of atherapeutically effective protocol for treatment of the patient. Theinitial and any subsequent dosages administered will depend upon thepatient's age, weight, condition, and the disease, disorder orbiological condition being treated. Depending on the therapeutic, thedosage and protocol for administration will vary, and the dosage willalso depend on the method of administration selected, for example, localor systemic administration. For a very potent peptoid, microgram (ug)amounts per kilogram of patient may be sufficient, for example, in therange of about 1 ug/kg to about 500 mg/kg of patient weight, and about100 ug/kg to about 5 mg/kg, and about 1 ug/kg to about 50 ug/kg, and,for example, about 10 ug/kg.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect, for each therapeutic,each administrative protocol, and administration to specific patientswill also be adjusted to within effective and safe ranges depending onthe patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient as well as peptoidpotency, severity of the disease being treated. For example, a dosageregimen of the peptoids can be oral administration of from 10 mg to 2000mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600 mg/day,in two to four (preferably two) divided doses, to reduce tumor growth.In a preferred embodiment, in cases where the peptoid is based on afused-ring cyclic benzocycloheptapyridine, the preferred dosage of theinhibitor is oral administration of from 50 to 600 mg/day, morepreferably 50 to 400 mg/day, in two divided doses. Intermittant therapy(e.g., one week out of three weeks or three out of four weeks) may alsobe used.

In one example of combination therapy in the treatment of pancreaticcancer, the peptoid is selected from Table 4 or. 5, administered orallyin a range of from 50 to 400 mg/day, in two divided doses, on acontinuous dosing regimen; and the antineoplastic agent is gemcitabineadministered at a dosage of from 750 to 1350 mg/m² weekly for three outof four weeks during the course of treatment. In another example ofcombination therapy in the treatment of lung cancer, the peptoid isselected from Table 4 or 5, administered orally in a range of from 50 to400 mg/day, in two divided doses, on a continuous dosing regimen; andthe antineoplastic agent is paclitaxel administered at a dosage of from65 to 175 mg/m² once every three weeks. In another example ofcombination therapy in the treatment of gliomas, the peptoid is selectedfrom Table 1, administered orally in a range of from 50 to 400 mg/day,in two divided doses; and the antineoplastic agent is temozolomideadministered at a dosage of from 100 to 250 mg/m². In another example ofcombination therapy, the peptoid is selected from Table 4 or 5,administered orally in a range of from 50 to 400 mg/day, in two divideddoses, on a continuous dosing regimen; and the antineoplastic agent is5-Fluorouracil (5-FU) administered either at a dosage of 500 mg/m² perweek (once a week), or at a dosage of 200–300 mg/m² per day in the caseof continuous infusion of the 5-FU. In the case of 5-FU administrationon a weekly injection, 5-FU may be administered in combination with afoliate agonist, e.g., Leucovoran (at a dosage of 20 mg/m² /week).

A preferred embodiment of the invention includes monitoring the effectsof the treatment with an AV peptoid for signs of tumor regression, andsubsequently adjusting the administration of further doses accordingly.For example, a person with breast carcinoma would be treated locallywith an agent such as cyclophosphamide methotrexate 5-FU (CMF) ortamoxifen or local radiation therapy and an AV peptoid. Subsequentmammography, ultrasound, or physical exams, as compared with the samepre-treatment tests, would direct the course and dosage of furthertreatment.

The attending clinician, in judging whether treatment is effective atthe dosage administered, will consider the general well-being of thepatient as well as more definite signs such as relief of disease-relatedsymptoms, inhibition of tumor growth, actual shrinkage of the tumor, orinhibition of metastasis. Size of the tumor can be measured by standardmethods such as radiological studies, and successive measurements can beused to judge whether or not growth of the tumor has been retarded oreven reversed. Relief of disease-related related symptoms such as pain,and improvement in overall condition can also be used to help judgeeffectiveness of treatment. Accordingly, preferred embodiments of theinvention include monitoring of the patient after treatment with an AVpeptoid for signs of tumor regression. Such monitoring includes but isnot limited to physical exam, CT scan, MRI, mammography, chest X-rays,bone scans, ultra-sounds, bronchoscopy, endoscopy, colonscopy,laparoscopy, and tests for tumor markers such as PSA, CEA, and CA125.The appropriateness of any form of monitoring will be determined by thenature of the cancer being treated.

EXAMPLES

Preparation of Monomers. Representative monomers of the invention,suitable for peptoid construction, were prepared as set forth below.Efforts have been made throughout the examples to insure accuracy withrespect to numbers used (e.g., amounts, temperature, pH, etc.) but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees centigrade, and pressure is at or near atmospheric.

Example 1 FMOC-N-Benzylglycine

A. Reaction 1

N-benzylglycine ethyl ester (Aldrich, 4.0 mL, 20.9 mmol) was dissolvedin methanol (40 mL) and treated overnight with aqueous KOH (10 M, 8 mL)at room temperature. TLC indicated complete conversion to product. Thesolution was cooled in an ice bath and carefully acidified to pH 2 withHCl. White crystals were collected and recrystallized from aqueousmethanol, to provide 3.95 g (93%) of the HCl salt.

B. Reaction 2

N-benzylglycine.HCl (1.07 g, 5.3 mmol) was dissolved in aqueousacetonitrile, and the pH brought to 9–10 with 1 N NaOH. A solution ofFMOC-Cl in acetonitrile was added dropwise, and the pH maintained byadding base, until the reaction was complete (as judged by TLC). The pHof the solution was lowered to 4, and the solution extracted with ethylacetate. The organic layer was washed with water and dried over sodiumsulfate. Silica gel chromatography (ethyl acetate/hexanes) yieldedFMOC-N-benzylglycine as an oil (1.43 g, 70%), which could berecrystallized from acetic acid/methanol. This monomer may be used inpeptoids at any position at which an aromatic side chain is desired.

Example 2 FMOC-N-isobutylglycine

A. Reaction 3

Isobutylamine (50 mL, 0.5 mol) was cooled in an ice bath, andbromoacetic acid (6.1 g, 43.9 mmol) added slowly as a solid, insuringthat each piece dissolved. After stirring overnight, the excess aminewas removed and MeOH was added to the resulting oil. The resultingmixture was concentrated, and repeated using MeOH/HCl. Finally, a whitesolid was recrystallized from ethanol/ether to provideN-isobutylglycine.HCl (3.95 g, 53.7%).

B. Reaction 4

N-isobutylglycine.HCl (1.25 g, 7.46 mmol) was dissolved in aqueousacetonitrile and treated with FMOC-Cl as described in part (A) above.After the reaction was complete, the pH was lowered to 2.5 and theaqueous solution extracted with ethyl acetate. The organic layer waswashed with water, saturated NaCl, and dried over sodium sulfate. Silicagel chromatography (ethyl acetate/hexanes) yieldedFMOC-N-isobutylglycine as an oil (1.29 g, 47%). Additional materialcould be recovered from impure chromatographic fractions. This monomermay be used in peptoids at any position at which an aliphatic,hydrophobic side chain is desired.

Example 3 FMOC-N-(N′-BOC-2-aminoethyl)glycine

A. Reaction 5

Ethylenediamine (65 mL, 0.97 mol) was cooled in an ice bath, andchloroacetic acid (10.0 g, 0.106 mol) added in small portions, allowingeach portion to dissolve. After the addition was complete, the solutionwas allowed to stir overnight at room temperature. Water was added andthe solution applied to a Dowex®-AG-1 column (OH⁻ form, 2.5×50 cm). Thecolumn was washed with water (2 L) until ninhydrin-negative, and theproduct eluted with 0.5 N HOAc. The ninhydrin-positive fractions werepooled, concentrated, and recrystallized from EtOH/Et₂ O/HCl to provideN-2-aminoethylglycine.HCl (9.7 g, 48%).

B. Reaction 6

N-2-aminoethylglycine.HCl (2.5 g, 13.1 mmol) was dissolved in water (20mL) and dioxane (25 mL). The pH was brought to 11.2 with concentratedNaOH. BOC-nitrophenylcarbonate (3.5 g, 14.6 mmol) was dissolved indioxane (20 mL) and added over 45 min with stirring, maintaining the pHwith a pH stat. After the addition, the solution was stirred for one dayat constant pH. Water was then added, the pH lowered to 6, and theresulting solution extracted with ether. The pH was further lowered to 3with KHSO₄ and extracted with ether, then lowered to 2 and extractedwith EtOAc. TLC indicated that the product remained in the aqueouslayer. The product was used without further purification.

The pH of the solution was adjusted to 9.5 with NaOH, providing a totalvolume of about 200 mL. Acetone (50 mL) was then added, andFMOC-N-hydroxysuccinimide (4.64 g, 13.6 mmol) in acetone (100 mL) addeddropwise while maintaining the pH. The reaction mixture was stirredovernight. The basic solution was extracted with ether and carefullyacidified to pH 2.5 with HCl and KHSO₄. The acidic solution was washedwith saturated NaCl and dried over sodium sulfate. After concentration,the solid was recrystallized from ethyl acetate/hexanes to provideFMOC-N-(N′-BOC-2-aminoethyl)glycine (4.74 g, 57% for two steps). Thismonomer may be used in peptoids at any position at which a basic sidechain is desired.

Example 4 FMOC-N-(2-t-butoxyethyl)glycine

A. Reaction 7

Ethanolamine (60 mL) was cooled in an ice bath, and chloroacetic acid(10.0 g, 10.5 mmol) added in portions, allowing each portion to fullydissolve. The solution was then heated to 60° C. for one hour. Aftercooling, the product was applied to a Dowex®-AG1 column (OH⁻ form,2.5×50 cm). The column was washed with water until the washes wereninhydrin-negative, and the product eluted with 0.5 N HOAc. Afterconcentration, N-2(hydroxyethyl) glycine (9.8 g, 52%) was obtained.

B. Reaction 8

N-2(hydroxyethyl) glycine (5.18 g, 28.9 mmol) was dissolved in 1 N NaOH(60 mL) and dioxane (60 mL). The pH was adjusted to 9.5 and the solutioncooled in an ice bath. FMOC-Cl (10.0 g, 38.7 mmol) in dioxane (50 mL)was added dropwise with stirring while maintaining the pH by addition ofNaOH. After the addition was complete, the solution was allowed to stirat room temperature for two more hours. The basic solution was extractedwith ether. Then, the solution was carefully acidified to pH 2.5 withHCl, and the acidic solution extracted with EtOAc, which was washed withNaCl and dried over sodium sulfate. After concentration, the product wasrecrystallized from ethyl acetate/hexanes to provideFMOC-N-hydroxyethylglycine (9.11 g, 92%).

C. Reaction 9

The product (805 mg, 2.36 mmol) was dissolved in MeOH and the solutionacidified with a few drops of H₂SO₄. The solution was heated at refluxfor 30 minutes, until TLC indicated a complete conversion to product.Water was added, and the solution extracted with ether, ethyl acetate,and methylene chloride. The ether and ethyl acetate solutions werecombined and washed with water and brine, and dried over sodium sulfate.The methylene chloride extract was washed with water and dried. Thecombined organic layers were concentrated to 880 mg of product, whichwas used without further purification. This product was dissolved inmethylene chloride (11 mL) and cooled in a dry ice bath. Isobutylene(about 10 mL) was added, followed by concentrated sulfuric acid (100μL). The flask was stoppered and allowed to stand at room temperature.After one week, the flask was cooled to −78° C., opened and allowed towarm up to room temperature under a stream of nitrogen. The methylenechloride solution was washed with sodium carbonate and water, and driedover sodium sulfate. The concentrated material was chromatographed usingethyl acetate/hexanes to provide two major products; by NMR, the desiredproduct and the bis t-butyl product.

The desired product (521 mg, 1.27 mmol) was suspended in 0.1 M sodiumphosphate (pH 8.0). Porcine liver esterase (100 μL, 108.5 u/mg, 10mg/mL) was added followed by Triton® (200 μL, 10% aqueous solution). ThepH was maintained at 8 by periodic addition of NaOH. After one day, thesolution was extracted with ethyl acetate. TLC indicated a slower movingcompound in addition to unreacted starting material, which was verifiedas FMOC-N-(2-t-butoxyethyl)glycine by NMR. This monomer may be used inpeptoids at any position at which a hydroxy-containing side chain isdesired.

Example 5 FMOC-N-carboxymethyl(t-butyl Ester)glycine

A. Reaction 10

Glycine, t-butyl ester (10.0 g, 52.3 mmol) was dissolved in water (150mL) and the pH adjusted to 9.5 with NaOH. Chloroacetic acid (1.1 g, 11.6mmol) in 50 mL water was added dropwise to the solution with stirringwhile maintaining the pH with a pH stat. After the addition wascomplete, the reaction was allowed to stir overnight. The basic solutionwas exhaustively extracted with EtOAc and CH₂ Cl₂ until there was noadditional glycine t-butyl ester in the aqueous layer, as judged by TLC.The material was used without further purification.

B. Reaction 11

The pH of the solution was adjusted to 9.5, and acetone (100 mL) added.A solution of FMOC-NHS (4.0 g, 11.9 mmol) in acetone (50 mL) was addedslowly and the pH maintained at 9.5. After stirring 2 days, the basicsolution was extracted with ether, cooled in an ice bath, and carefullyacidified to pH 2.5 with KHSO₄. The acidic solution was extracted withethyl acetate. The organic layer was washed with water and dried oversodium sulfate. After concentration, FMOC-N-carboxymethyl(t-butylester)glycine (3.07 g, 64% from chloroacetic acid) was obtained as anoil. This monomer may be used in peptoids at any position at which anacidic side chain is desired.

Example 6 Preparation of Peptoids

Di- and tri-peptoids containing 1–3 N-substituted glycine analogs of theinvention were prepared using the N-substituted amino acidsFMOC-N-isobutylglycine (Leu*) and FMOC-N-benzylglycine (Phe*). The aminoacids were loaded onto a Wang resin (S.-S. Wang, J Am Chem Soc (1973)95:1328) and coupled usingbenzotriazoyloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) and diisopropylethylamine (DIEA). Substitution levels weredetermined using standard analytical procedures by quantifying theamount of FMOC released by treatment with piperidine in DMF. Theseresins are routinely capped with benzoyl chloride/pyridine prior tofurther coupling reactions.

HPLC analysis was performed using a Hewlett-Packard Diode-Array 1090Liquid Chromatograph, using a 2% gradient of 0–100% acetonitrile (0.1%trifluoroacetic acid)/H₂O (0.1% TFA) over 50 minutes, with an initial 5min delay (flow rate 0.8 mL/min). The column used was a 40 mm××25 cmVydac® C-18 stainless steel column. IR spectra were obtained using aNicolet FT-IR. FMOC-amino acids, loaded resin, and BOP were obtainedfrom Advanced Chemtech.

(A) FMOC-Leu*-Cl was prepared by dissolving FMOC-N-isobutylglycine (150mg, 0.42 mmol) in CH₂ Cl₂ (2.8 mL), and adding thionyl chloride (309 μL,4.2 mmol) and 3 μL DMF (0.04 mmol). The reaction mixture was stirred for5 hours and concentrated in vacuo, repeatedly dissolving in CH₂ Cl₂(3××) to provide FMOC-Leu*-Cl as a colorless oil (146 mg, 92%): IR(cm⁻¹) -1804, 1720, 1715. FMOC-Phe*-Cl was similarly prepared.

(B) The Wang resin (0.94 mmol/g, Applied Biosystems, Inc.) was loaded bycombining FMOC-Phe* (0.8 mmol) with Wang resin (325 mg, 0.3 mmol) in CH₂Cl₂ (4.5 mL). BOP (267 mg, 0.6 mmol) was added and dissolved, followedby DIEA (265 μL, 1.5 mmol). The resulting slurry was stirred at 23° C.for 24 hours. The resin was then filtered and washed with CH₂ Cl₂ and40% MeOH/CH₂ Cl₂, and dried under vacuum. The resin was then capped byswelling with CH₂ Cl₂ (4.3 mL) and cooled to 0° C. Pyridine (120 μL) wasadded followed by benzoyl chloride (140 μL). The resin was stirred whilewarming to 23° C. over 1 hour, filtered and washed with CH₂ Cl₂ anddried under vacuum to provide FMOC-Phe*-Wang.

(C) Similarly, proceeding as in part (B) above but substitutingFMOC-N-isobutylglycine, FMOC-N-(N′-BOC-2-aminoethyl)glycine,FMOC-N-(2-t-butoxyethyl)glycine, or FMOC-N-carboxymethyl(t-butylester)glycine for FMOC-N-benzylglycine, the corresponding resins areprepared.

(D) FMOC-Phe*-Wang resin (63 mg, 0.43 mmol/g) was deprotected by initialwashing with 20% piperidine in DMF, followed by treatment for 20 min.After repeatedly washing with DMF, MeOH, and CH₂ Cl₂, the resin wastreated with FMOC-Leu*-Cl (30 mg, 0.08 mmol) in CH₂ Cl₂ (380 μL).Pyridine (110 μL, 1.4 mmol) was added, and the resin shaken for 4 hours.Monitoring the coupling (A. Grandas et al., Int J Peptide Protein Res(1989) 33.386–90) revealed that the reaction was complete within 20 min.Filtration and washing with CH₂ Cl₂ and MeOH provided the fully coupledresin FMOC-Leu*-Phe*-Wang.

(E) Similarly, proceeding as in parts (B–D) above, the following resins(and their deprotected forms) were prepared:

-   FMOC-Leu*-Pro-Wang; FMOC-Leu*-Leu*-Wang; FMOC-Leu*-Leu-Wang;    FMOC-Phe*-Phe*-Wang; FMOC-Gly-Leu*-Wang; FMOC-Leu*-Phe-Wang; and    FMOC-Leu*-Leu*-Phe-Wang.

Leu* indicates N-isobutylglycine, and Phe* indicates N-benzylglycine.The peptoids are cleaved from the resins using 95% TFA, while thereaction was monitored by RP-HPLC as described above. The results areshown in Table 3 as follows:

TABLE 3 Peptoids and Retention Times Peptoid Retention Time (min)FMOC-Leu* 38.7 Leu* (not retained) FMOC-Phe* 39.3 Phe* 15.0FMOC-Leu*-Leu* 40.1 Leu*-Leu* 19.7 FMOC-Leu*-Phe* 41.1 FMOC-Phe*-Phe*43.4 FMOC-Leu*-Leu 40.4 Leu*-Leu 21.9

Example 7 Solid Phase Chemistry

Resins: Unloaded WANG (HMP) resin, Rink resin, and preloaded PAM resinswere bought from Advanced Chemtech or ABI. The first amino acid wascoupled to the WANG and Rink resins using the PyBrop method describedbelow.

Resin Deprotection: The resin was treated with a 20% piperidine in DMFsolution for one minute, drained, and repeated for 20 m. After draining,the resin was washed with DMF 3 times and methylene chloride 5–7 times.

Substitution Level: A preweighed dried amount of resin is treated with asolution of 20% piperidine in DMF (300λ⁻¹ mL) in an eppendorf tube on avortexer. After 20–30 m, the tubes are centrifuged for a few minutes tosettle the resin. An aliquot is removed (50λ) and diluted to 1 mL withacetonitrile. The absorbance at 300 nm is recorded vs. a standard of thesame dilution pip/DMF in ACN. In general, the spectrum from 280–320 nmis taken to ensure the characteristic pattern for an FMOC derivedproduct. The following formula is used to calculate the mmol/g(substitution level) of the resin: mmol/g=[Abs(300 nm)][λpip/DMFsolution][ACN dilution]/[mg resin][εM⁻¹ cm⁻¹][1 cm], e.g., 1.47 mgresin, A₃₀₀=0.298, 0.5 mL pip/DMF used, 50/1000 dilution.

Coupling: Acyl halide chemistry: The amino acid (1 mmol) was dissolvedin methylene chloride (10 mL) and treated with thionyl chloride (750λ)and DMF (10λ). The reaction was monitored by TLC or HPLC by mixing 10λwith 100λ methanol. In all cases studied, the R_(f) of the methyl esterwas greater than the corresponding R_(f) of the carboxylic acid. Afterstirring 30 m to 2 h, the solution was concentrated on a rotavap severaltimes from methylene chloride, and benzene or toluene, followed by highvacuum concentration to remove the solvents. In general, the compoundswere oils, and often colored yellow to brown. They were used withoutfurther purification. The acyl halide was dissolved in an appropriateamount of methylene chloride and was added to the deprotected resin,followed by a solution of DIEA/methylene chloride. After an appropriatetime, usually 1 hour, the resin was drained and washed well withmethylene chloride, dried, and assayed for FMOC content.

Coupling: PyBroP chemistry: The resin (˜100 mg, 0.05 mmol), afterdeprotection, was washed well (5–10 times) with methylene chloride. A0.2 mmol aliquot of the amino acid was weighted into a vial. This wasdissolved in 1.0 mL methylene chloride and 0.1 mL DIEA. A 0.2 mmolaliquot of PyBroP (bromo-tris-pyrrolidino-phosphoniumhexafluoro-phosphate), purchased from Nova chemicals UK, was weightedinto another vial. It was also dissolved in 1.0 mL methylene chloride,however, this solution remained cloudy. The amino acid solution wasadded to the resin followed by the PyBroP solution. The mini-column wascapped, vented, and gently shaken for one hour. The resin was drained,washed well with methylene chloride and ready for a repeat coupling orcapping.

Coupling: DIC/HOBt chemistry: The resin (˜50 mg, 0.033 mmol), afterdeprotection, was washed well (5–10 times) with DMF. To the resin wasadded 440λ of DMF followed by 170λ of a 1M solution of amino acid in DMFand 170λ. of a 1M solution of DIC and HOBt in DMF. The mini-column wascapped, vented, and gently shaken for one hour. The resin was drained,washed well with methylene chloride and ready for a repeat coupling orcapping.

Capping: After coupling, the resin was washed well with methylenechloride and treated with 100λ acetic anhydride, 100λ pyridine, and 2 mLmethylene chloride for 30 minutes.

Example 8 Polymer Purification and Analysis

Polymers using WANG Resin: Polymers prepared using the WANG or RINKresins were cleaved using the standard protocol, i.e., 95% aqueous TFAfor one hour at room temperature. No scavengers were necessary (no Trp,Tyr, Met). The TFA solution was filtered, dried down, and resuspended in20% aqueous acetic acid for HPLC analysis.

Polymers using PAM Resin: Resins were cleaved in HF at 0° C. for onehour. Either the resin was extracted with DMF, or the resin wasextracted with 50% aqueous acetic acid, concentrated, and redissolved inacetonitrile for HPLC analysis. In both cases, the organic solvent wasdiluted to approximately 25% with water before reversed phase HPLC onC-18 support.

The following examples are used to assay for the ability of AV peptoidsto interact with and preferably inhibit the activity of an Inhibitor ofApoptosis protein (IAP), as measured by IAP binding, procaspase-3activation or promotion of apoptosis. Note that these assays may be usedto assay peptoids for requisite IAP binding as well as to screen foragents (e.g. antagonists) which potentiate peptoid-IAP binding. The invitro binding and FP assays (Examples 9–11) are particularly suited toidentify peptoid antagonists, i.e. compounds which competitivelydisplace the peptoid, preferably at at least 1%, more preferably at atleast 10%, more preferably at at least 50%, and most preferably inexcess, of equimolar displacement. Accordingly, the invention providessuch AV peptoid antagonists produced (i.e. initially identified through)by these methods.

Example 9 In vitro IAP (BIR) Binding/Interaction Assay

Interaction between AV peptoids and IAPs was examined by GST-mediatedpull-down assays. Approximately 0.4 mg of a recombinant IAP fragment(second BIR (baculoviral IAP repeat) motif of XIAP) was bound to 200 mlof glutathione resin as a GST-fusion protein and incubated with 0.5 mgof radiolabeled AV peptoids at room temperature. After extensive washingwith an assay buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, and 2mM dithiothreitol (DTT), the complex was eluted with 5 mM reducedglutathione and visualized by SDS-PAGE with Coomassie staining. AVpeptoids are shown to specifically bind the IAP.

In particular experiments, we demonstrate that wild-type N-terminal Smacfragments are sufficient to effect IAP binding. Smac stimulatesactivation of procaspase-3 by relieving the IAP inhibition^(20, 21). Allmembers of the IAP family contain at least one BIR (baculoviral IAPrepeat) motif and many contain three⁹. Recent experiments indicate thatdifferent BIR domains may exhibit distinct functions; for example, thesecond BIR domain (BIR2) of XIAP appears to be a potent suppressor ofapoptosis and a direct inhibitor for caspases whereas neither BIR1 norBIR3 exhibited similar activity²³. We report here that Smac andN-terminal fragments thereof can specifically interact with the secondor third BIR domain of XIAP. Similar results are obtained withN-terminal fragments of Smac homologs, Reaper, Grim, and Hid, as well assynthetic homologs; see Table 4.

TABLE 4 IAP binding of AV peptoids comprising wild-type Smac, Reaper,Grim and Hid N-terminal peptide sequences and synthetic homologs.. IAPAV Peptoid Binding Smac-2: NH2-AV—COOH ++ Smac-3: NH2-AVP—COOH +++Smac-4: NH2-AVPI—COOH (SEQ ID NO: 5) +++ Smac-5: NH2-AVPIA-COOH (SEQ IDNO: 6) +++ Smac-6: NH2-AVPIAQ-COOH (SEQ ID NO: 7) +++ Smac-7:NH2-AVPIAQK—COOH (SEQ ID NO: 8) +++ Smac-7R: NH2-KQAIPVA-COOH (SEQ IDNO: 9) 0 Reaper-3: NH2-AVA-COOH +++ Reaper-4: NH2-AVAF—COOH (SEQ ID NO:10) +++ Reaper-5: NH2-AVAFY—COOH (SEQ ID NO: 11) +++ Reaper-6:NH2-AVAFYI—COOH (SEQ ID NO: 12) +++ Grim-3: NH2-AIA-COOH +++ Grim-4:NH2-AIAY—COOH (SEQ ID NO: 13) +++ Grim-5: NH2-AIAYF—COOH (SEQ ID NO: 14)+++ Hid-4: NH2-AVPF—COOH (SEQ ID NO: 15) +++ Hid-5: NH2-AVAFY—COOH (SEQID NO: 11) +++ Hid-6: NH2-AVAFYL-COOH (SEQ ID NO: 16) +++ Synth-3A:NH2-AIP—COOH +++ Synth-3B: NH2-ALP—COOH +++ Synth-3C: NH2-ALA-COOH +++Synth-3D: NH2-ALV—COOH +++ Synth-3E: NH2-AVV—COOH ++

Example 10 High-Throughput In Vitro Fluorescence Polarization BindingAssay

-   Sensor: Rhodamine-labeled AV peptoid (final conc.=1–5 nM)-   Receptor: Glutathione-S-transferase/BIR fusion protein (final    conc.=100–200 nM)-   Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6-   1. Add 90 microliters of peptoid/BIR mixture to each well of a    96-well microtiter plate.-   2. Add 10 microliters of test compound per well.-   3. Shake 5 min and within 5 minutes determine amount of fluorescence    polarization by using a Fluorolite FPM-2 Fluorescence Polarization    Microtiter System (Dynatech Laboratories, Inc).    Tested AV peptoids and Smac fragments (except 7R) significantly and    specifically bind the IAP BIR domain, see Table 5 as follows:

TABLE 5 AV Peptoids Effecting IAP Binding and Test CompoundsDemonstrating Peptoid Displacement (Antagonists). IAP Test Compounds**Dis- AV Peptoid* Binding demonstrating displacement placement XWBC2411+++ N67218, N63439, S25539, +++ S57739, S43643 XWBC0093 +++ N32665,N19093, N37883, +++ S38674, S03480 CDWB4558 +++ C32829, C10438, C85376,+++ C26347, C46792 WYRMS7726 +++ N47639, N27374, S29387, +++ S68093,C62039 WYRMS1592 +++ S89374, S26293, S10328, +++ S72934, C39384WYRMS9311 +++ N28474, S26743, C29287, +++ C27478, C32937 TOKA0283 +++S29374, S07984, S28340, +++ S14242, C86908 TOKA6305 +++ N16261, S21652,S15533, +++ S46658, S93399 TOKA3375 +++ S37355, S79685, S76246, +++S97421, S14243 BAKHC7539 +++ S32532, C09485, C36389, +++ C54200, C15437BAKHC2294 +++ N36536, N94777, S32520, +++ S36378, C21233 BAKHC5275 +++S59273, S32688, S29875, +++ S78993, C20357 *Mimetics with BCdesignations are made by the backbone cyclic proteinomimetic protocol(Kasher et al., 1999, supra); mimetics with RMS designations are made bythe rigid molecular scaffold protocol (Andrade-Gordon et al., 1999,supra); mimetics with KA and KHC designations are made by theα-ketoamide and α-ketoheterocyycle protocols (Boatman et al., 1999,supra), respectively. **Test compounds with N, C and S designations areobtained from natural (Merck & Co. Inc.), combinatorial (DuPontPharmaceuticals Co.) and other synthetic libraries (Pangea Systems),respectively.

-   11. High throughput solid phase peptoid-BIR    binding/binding-interference assay.-   A. Reagents:    -   Neutralite Avidin: 20 μg/ml in PBS.    -   Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room        temperature.    -   Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1%        glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA,        cocktail of protease inhibitors.    -   ³³P Smac peptoid 10×stock: 10⁻⁸–10⁻⁶M “cold” peptide mimetic        supplemented with 200,000–250,000 cpm of labeled mimetic        (Beckman counter). Place in the 4° C. microfridge during        screening.    -   Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor        (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine        (Sigma # B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF        (BMB #917575), and 2mM NaVO₃ (Sigma # S-6508) in 10 ml of PBS.    -   BIR: 10⁻⁷–10⁻⁵ M biotinylated BIR domain (supra) in PBS.-   B. Preparation of assay plates:    -   Coat with 120 μl of stock N-Avidin per well overnight at 4° C.    -   Wash 2 times with 200 μl PBS.    -   Block with 150 μl of blocking buffer.    -   Wash 2 times with 200 μl PBS.-   C. Assay:    -   Add 40 μl assay buffer/well.    -   Add 10 μl compound or extract.    -   Add 10 μl ³³P-peptoid (20–25,000 cpm/0.1–10        pmoles/well=10⁻⁹–10⁻⁷ M final conc).    -   Shake at 25° C. for 15 minutes.    -   Incubate additional 45 minutes at 25° C.    -   Add 40 μM biotinylated BIR (0.1–10 pmoles/40 ul in assay buffer)    -   Incubate 1 hour at room temperature.    -   Stop the reaction by washing 4 times with 200 μM PBS.    -   Add 150 μM scintillation cocktail.    -   Count in Topcount.-   D. Controls for all assays (located on each plate):    -   a. Non-specific binding    -   b. Soluble (non-biotinylated BIR) at 80% inhibition        AV peptoids and Smac fragments (except 7R) significantly and        specifically bind the IAP BIR domain.

Example 12 Hela Cell Extracts: Radiolabeled Procaspase-3 ActivationAssay

20 mg S-100 extracts of HeLa cells were incubated alone (Control), orwith AV peptoids (50 nM) nM, or with 30–1000 mM of N-terminal Smacpeptides in different lengths. The reactions were carried out with theaddition of 1 mM DATP, 1 mM additional MgCl₂, 0.2 mg/ml horse heartcytochrome c, and 1 ml of in vitro translated, ³⁵S-labeled caspase-3 ina final volume of 20 ml. The reaction mixtures were incubated at 30° C.for 1 hr followed by electrophoresis on a 15% PAGE gel. The gel wassubsequently transferred onto a nitrocellulose filter and exposed to aphosphoimaging cassette. All AV peptoids and Smac fragmentssignificantly promoted activation of procaspase-3, whereas negativecontrol Smac-7R did not.

Example 13 Hela Cell Extracts: Spectrofluorometric Procaspase-3Activation Assay

Human Hela S3 cells were cultured in 150-mm tissue culture dishes inDMEM medium (Dulbecco's modified eagle's medium containing 100 U/ml ofpenicillin and 100 ug/ml of streptomycin sulfate) supplemented with 10%(v/v) fetal calf serum, and grown in monolayer at 37° C. in anatmosphere of 5% CO₂. Cells at 70% confluence were washed once with1×phosphate-buffered saline (PBS) and harvested by centrifugation at800×g for 5 min at 4° C. The cell pellets were resuspended in 3 volumeof Buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCL, 1.5 mM MgCl2, 1 mMsodium EDTA, 1 mM sodium EGTA, 1 mM DTT, and 0.1 mM PMSF) and cellextracts were prepared as described in Liu et al. (24). Human c-IAP-1 orc-IAP-2, or XIAP either full length of truncated proteins that containthe first three BIR domain, or the second, or third BIR domain are addedto the HeLa cell extracts and the caspase activation reaction is startedby adding 1 mM dATP and 300 nM cytochrome c. The caspase-3 activity ismeasured by spectrofluorometric assay as previously described byMacFarlane et al. (1997, J. Cell Biol. 137, 469–479). Aliquots of 8 mgof S-100 prepared as in Liu et al. were assayed in 96-well microtiterformat in a 150 ml of reaction containing 0.1 mM Hepes, PH 7.4, 2 mMDTT, 0.1% (w/v) Chaps, and 1% (w/v) Sucrose. The reactions were startedby adding caspase specific fluorogenic substratebenzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin-Z-DEVD-AFC (commercially available from Enzyme Systems) to the finalconcentration of 20 mM and continued at 37° C. for 30 min. Liberation ofAFC from the substrates was monitored continuously usingexcitation/emission wavelenth paires of 400/505 nm. AV peptoids and Smacfragments (except 7R) significantly promoted activation of procaspase-3.

Example 14 Reconstituted Recombinant Radiolabeled Procaspase-3Activation Assay

AV peptoids and N-terminal Smac peptides (30–3000 mM) were incubatedwith recombinant human Apaf-1 (40 nM), recombinant human procaspase-9 (2nM), purified horse heart cytochrome c (nM) and mouse XIAP (70 nM) inthe presence of 1 mM dATP, 1 mM MgCl₂ and 1 ml of in vitro translated,³⁵S-labeled caspase-3 in a final volume of 20 ml. The reaction mixtureswere incubated at 30° C. for 1 hr followed by electrophoresis on a 15%PAGE gel. The gel was transferred onto a nitrocellulose filter andexposed to a phosphoimaging cassette. Active Smac protein (50 nM) and aninactive peptide Smac-7R (3000 mM) are also included as controls. All AVpeptoids and Smac fragments (except 7R) significantly promotedactivation of procaspase-3.

Example 15 Reconstituted Recombinant Spectrofluorometric Procaspase-3Activation Assay

A reconstituted recombinant procaspase-3 activation system isconstructed as described above except the human caspase-3 is producedfrom bacterial expression as described in Liu et al., 1997 (supra) andis not labeled. The caspase-3 activity is measured byspectrofluorometric assay as previously described by MacFarlane et al.(1997, J. Cell Biol. 137, 469–479). Aliquots of 8 mg of S-100 preparedas above were assayed in 96-well microtiter format in a 150 ml ofreaction containing 0.1 mM Hepes, PH 7.4, 2 mM DTT, 0.1% (w/v) Chaps,and 1% (w/v) Sucrose. The reactions were started by adding caspasespecific fluorogenic substratebenzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin-Z-DEVD-AFC (SEQ ID NO:17) (commercially available from Enzyme Systems) to thefinal concentration of 20 mM and continued at 37° C. for 30 min.Liberation of AFC from the substrates was monitored continuously usingexcitation/emission wavelenth paires of 400/505 nm. AV peptoids and Smacfragments (except 7R) significantly promoted activation of procaspase-3.

Example 16 Cell-based Assay: Smac Peptides Potentiate Apoptosis Inducedby UV or Etoposide in Cultured HeLa Cells

0.75×105 of HeLa-S cells/well were plated in 48-well tissue cultureplate. Cells were incubated with 1 mM inactive Smac peptide (Smac-7R,NH2-KQAIPVA-COOH, SEQ ID NO:9) or with 1 mM N-terminal 4-amino acid Smacpeptide (Smac-4, NH2-AVPI-COOH, SEQ ID NO:5), with selected AV peptoidscomprising peptide mimetics, or with vehicle only (Control) for 12 hr.The cells were then treated with either 320,000 microjoules of UVirradiation using a Stratalinker or with 100 mM chemotherapeuticEtoposide. Cells were then stained with 1 mg/ml Hoechst 33342 dye atdifferent time points and apoptotic cells were counted as those withcondensed nuclear chromatin under a fluorescent microscopy. AV peptoids,including Smac peptides, showed significant increases in apoptoticinduction at 2, 4 and 6 hrs (for UV insult) and at 10 and 20 hr (foretoposide); see, e.g. Table 6, below.

TABLE 6 AV Peptoids and AV peptoid Antagonists (Example 10) Shown toEnhance Apoptosis. AV Peptoid Apoptosis Antagonists Apoptosis XWBC2411+++ N67218, N63439, S25539, +++ S57739, S43643 XWBC0093 +++ N32665,N19093, N37883, +++ S38674, S03480 CDWB4558 +++ C32829, C10438, C85376,+++ C26347, C46792 WYRMS7726 +++ N47639, N27374, S29387, +++ S68093,C62039 WYRMS1592 +++ S89374, S26293, S10328, +++ S72934, C39384WYRMS9311 +++ N28474, S26743, C29287, +++ C27478, C32937 TOKA0283 +++S29374, S07984, S28340, +++ S14242, C86908 TOKA6305 +++ N16261, S21652,S15533, +++ S46658, S93399 TOKA3375 +++ S37355, S79685, S76246, +++S97421, S14243 BAKHC7539 +++ S32532, C09485, C36389, +++ C54200, C15437BAKHC2294 +++ N36536, N94777, S32520, +++ S36378, C21233 BAKHC5275 +++S59273, S32688, S29875, +++ S78993, C20357

Example 17 In Vivo Metastasis Assay

Immunosuppressed mice (athymic nude/nude SCID females from HarlanSprague Dawley) are housed in autoclaved cages with microisolator tops,and all manipulations of the animals are done in a laminar flow hoodafter wiping down both the hood, gloves and cages with ABQ sterilant.The mice are fed sterile Pico Lab Chow (Purina) and autoclaved St. Louistap water. AV peptoids are administered intra-gastrically daily to themice in sterile water containing 2% carboxymethyl cellulose via sterile,disposable animal feeding needles (Poper & Sons Cat #9921; 20 g×1.5″),seven days a week between 7:00 and 8:00 am. The compounds and control(sterile water plus 2% carboxymethyl cellulose) are kept stored at −80°C. wrapped in aluminum foil to prevent any light induced changes, andeach day's supply is thawed just prior to use.

Compounds are tested for their effects on the metastatic potential ofC8161 cells injected intravenously via the tail vein: at 40 and 100mg/kg, compared to the control. The concentration of the compounds inthe vials used to give the 100 mg/kg doses are 2.5 times that in the 40mg/kg dose so that approximately the same volume is used in both cases,approximately 0.5 mL/animal. The experiments start with nine animals pergroup at day −4. On day zero, 2×10⁵ C8161 cells in cold Hank's BalancedSalt Solution (HBSS) are injected intravenously via tail veininoculation. The protocol is continued for an additional 24 days, atwhich time the animals are sacrificed and their lungs removed and fixedin a solution of Bouins/formaldehyde (5 parts: 1 part). Tumors arequantified on the entire surface of the lungs by rotating the lungs andcounting the tumors on each lobe using a 6× magnifying glass.Statistical analysis is performed using the statistical package ofMicrosoft's Excel spreadsheet software.

The effects of test AV peptoid, at two different concentrations, on themetastatic potential of C8161 cells in SCID mice are evaluated: oralgavaging of the animals with tested AV peptoids significantly reducesthe number of lung metastases in the SCID mouse population.

Example 18 In Vivo Combination Therapy: B.I.D. & Q.I.D

The effect of in vivo combination therapy of AV peptoids (20 or 80mpk/dosing p.o. or i.p.) with chemotherapies paclitaxel (5 or 20 mpk),5-Fu (50 mpk), vincristine (1 mpk) or cytoxan (100 mpk, BID, ip) on HTB177 xenografts (NCI-H460, a human lung large cell carcinoma) using twoand four times a day dosing is demonstrated in athymic nu/nu femalemice, 5–6 weeks old. On Day 0, HTB 177 cells, 3×10⁶, are injected s.c.into the flank of 220 mice and the mice divided into treatment andcontrol groups:

Peptoid was dissolved in 20% hydroxyl-propyl-betacyclodexatrin (VehicleI); 0.2 ml of peptoid solution was the dosing volume. Paclitaxel wasdissolved in a diluted ethanol/cremophor EL solution (Vehicle II) andthe i.p. dosing volume for paclitaxel was 0.1 ml. Cytoxan, 5-FU, andVincristine were dissolved in sterile water. The 80 mpk dosing peptoidsolution is made by adding 17 ml of 20% HPBCD to a 50 ml tube containing136 mg of peptoid to dissolve. The mixture was sonicated until acomplete solution was made. The 20 mpk dosing solution is made byplacing 2 ml of the 80 mpk solution into a 15 ml tube, adding 6 ml of20% HPBCD, and vortexing the solution to mix it.

Tumor cells are inoculated into mice in the morning of Day 0, and themice weighed, randomized, and ear-marked afterwards. Drug treatmentbegins at 7:30 am on Day 4. The animals are dosed with peptoid orvehicle I solution, at 7:30 am, and 7:30 pm, 7 days a week. Tumor growthis quantitated by measuring tumor volume on Day 7 and Day 14. Bothpeptoid and chemotherapies demonstrate inhibition; combination therapiesprovide enhanced inhibition over either therapy alone.

Example 19 In Vivo Therapy in the WAP-RAS Transgenic Model

Peptoid and Paclitaxel combination efficacy is also evaluated in theWap-ras transgenic model. This model is used in a therapeutic mode inwhich treatments are initiated after mice had well developed tumors.

Peptoid (20 mpk/dosing po) was dissolved in 20%hydroxyl-propyl-betacyclodexatrin (Vehicle I). 0.2 ml of peptoidsolution is the oral dosing volume. Paclitaxel (5 mpk/dosing ip) wasdissolved in a diluted ethanol/cremophor EL solution (vehicle II) andthe i.p. dosing volume for paclitaxel was 0.1 ml.

The mice are weighed, randomized, and ear-marked on Day 0. Peptoidtreatment and Vehicle I treatment began on Day 1 and continued every 12hours until Day 21. Paclitaxel and Vehicle II treatments are started onDay 4 and continued daily on Day 5, 6, and 7. Wap-ras tumors do notrespond to treatment with Paclitaxel but do respond to peptoid treatmentat 20 mpk alone and combined therapy enhanced efficacy.

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All publications and patent applications cited in this specification andall references cited therein are herein incorporated by reference as ifeach individual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of screening for an agent which modulates the interaction ofan AV peptoid with an Inhibitor of Apoptosis Protein (IAP), said methodcomprising the steps of: incubating a mixture comprising: an AV peptoid,wherein the AV peptoid is a peptide comprising AX₁, wherein X₁ is V, Ior L, or a peptide mimetic thereof, which interacts with an IAP asmeasured by IAP binding, procaspase-3 activation or promotion ofapoptosis, a second baculoviral IAP repeat domain (BIR2) of XIAP, acandidate agent; under conditions whereby, but for the presence of saidagent, the peptoid specifically interacts with the BIR2 at a referenceaffinity; detecting a specific interaction of the peptoid with the BIR2to determine an agent-biased affinity, wherein a difference between theagent-biased affinity and the reference affinity indicates that theagent modulates the interaction of the peptoid to the BIR2 of the XIAP.2. The method of claim 1, wherein the detecting step comprises measuringin vitro binding of the peptoid to the BIR2 by pull-down assay,fluorescent polarization assay or solid-phase binding assay.
 3. Themethod of claim 1, wherein the mixture further comprises procaspase-3and a caspase-3 substrate and the detecting step comprises measuring theinteraction inferentially by detecting a reaction product of thecaspase-3 substrate and caspase-3 generated by activation of theprocaspase-3.
 4. The method of claim 1, wherein the AV peptoid is apeptide comprising AX₁X₂, wherein X₁ is V, I or L and X₂ is P or A. 5.The method of claim 1, wherein the AV peptoid is a peptide comprisingAX₁X₂, wherein X₁ is V and X₂ is P.
 6. The method of claim 1, whereinthe AV peptoid comprises the amino acid sequence Ala-Val-Pro, is fewerthan 20 residues in length, has a molecular weight less than 1000, andinteracts with an Inhibitor of Apoptosis protein (IAP) as measured byTAP binding.
 7. The method of claim 6, wherein the peptoid has amolecular weight less than
 500. 8. The method of claim 6, wherein thepeptoid is 3 to 5 residues in length.
 9. The method of claim 6, whereinthe peptoid is 3 residues in length.
 10. The method of claim 6, whereinthe peptoid comprises a carbon-carbon or acyl bond in place of a peptidebond.
 11. The method of claim 6, wherein the peptoid comprises anamino-terminal or carboxyl terminal blocking group selected from thegroup consisting of: t-butyloxycarbonyl, acetyl, alkyl, succinyl,methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl,fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl,and 2,4,-dinitrophenyl.
 12. The method of claim 6, wherein the peptoidcomprises an N-terminus and a C-terminus and peptidyl bonds, wherein oneor more of the peptidyl [—C(O)NR—] linkages have been replaced by anon-peptidyl linkage selected from the group consisting of: a—CH₂-carbamate linkage [—CH₂—OC(O)NR—]; a phosphonate linkage; a—CH₂-sulfonamide [—CH₂—S(O)₂NR—] linkage; a urea [—NHC(O)NH—] linkage; a—CH₂-secondary amine linkage; or an alkylated peptidyl linkage[—C(O)NR⁶—] where R⁶ is lower alkyl.
 13. The method of claim 6, whereinthe peptoid comprises an N-terminus and a C-terminus and peptidyl bonds,wherein the N-terminus is derivatized to a group selected from the groupconsisting of: a —NRR¹ group; a —NRC(O)R group; a —NRC(O)OR group; a—NRS(O)₂R group; a —NHC(O)NHR group, where R and R¹ are hydrogen orlower alkyl with the proviso that R and R¹ are not both hydrogen; to asuccinimide group; a benzyloxycarbonyl-NH—(CBZ-NH—) group; and abenzyloxycarbonyl-NE- group having from 1 to 3 substituents on thephenyl ring selected from the group consisting of lower alkyl, loweralkoxy, chloro, and bromo.
 14. The method of claim 6, wherein thepeptoid comprises an N-terminus and a C-terminus and peptidyl bonds,wherein the C terminus is derivatized to —C(O)R² where R² is selectedfrom the group consisting of lower alkoxy, and —NR³R⁴ where R³ and R⁴are independently selected from the group consisting of hydrogen andlower alkyl.
 15. The method of claim 6, wherein the peptoid comprises anN-terminus and a C-terminus and peptidyl bonds wherein: one or more ofthe —C(O)NH— linkage have been replaced by a linkage selected from thegroup consisting of a —CR₂OC(O)NR-linkage; a phosphonate linkage; a —CH₂S(O)₂ NR— linkage; a —CH₂ NR— linkage; and a —C(O)NR⁶-linkage, and a—NHC(O)NH— linkage where R is hydrogen or lower alkyl and R⁶ is loweralkyl, and the N-terminus is selected from the group consisting of a—NRR¹ group; a —NRC(O)R group; a —NRC(O)OR group; a —NRS(O)₂R group; a—NHC(O)NHR group; a succinimide group; a benzyloxycarbonyl-NH— group;and a benzyloxycarbonyl-NH— group having from 1 to 3 substituents on thephenyl ring selected from the group consisting of lower alkyl, loweralkoxy, chloro, and bromo, where R and R¹ are independently selectedfrom the group consisting of hydrogen and lower alkyl, and theC-terminus has the formula —C(O)R² where R² is selected from the groupconsisting of hydroxy, lower alkoxy, and —NR³R⁴ where R³ and R⁴ areindependently selected from the group consisting of hydrogen and loweralkyl and where the nitrogen atom of the —NR³R⁴ group can optionally bethe amine group of the N-terminus of the peptide so as to form a cyclicpeptide, and physiologically acceptable salts thereof.
 16. The method ofclaim 6, wherein the peptoid further comprises an N-substituted glycineanalog having the general formula I: X_(n)NRCH₂COOX_(c), wherein R is anamino acid α-substituent and the radicals X_(n) and X_(c) are eitherchains of conventional amino acids, chains of one or more N-substitutedglycine analogs, or chains in which conventional amino acids andN-substituted glycine analogs are interspersed.
 17. The method of claim6, wherein the peptoid further comprises an N-substituted glycine analoghaving the general formula I: X_(n)NRCH₂COOX_(c), wherein R is an aminoacid α-substituent and the radicals X_(n) and X_(c) are either chains ofconventional amino acids, chains of one or more N-substituted glycineanalogs, or chains in which conventional amino acids and N-substitutedglycine analogs are interspersed, wherein the N-substituted glycineanalogs are selected from the group consisting of glycine analogs inwhich R is ethyl, prop-1-yl, prop-2-yl, 1-methylprop-1-yl,2-methylprop-1-yl, benzyl, 4-hydroxybenzyl, 2-hydroxyethyl,mercaptoethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,2-methylthioeth-1-yl, carboxymethyl, 2-carboxyethyl, carbamylmethyl,2-carbamylethyl, 3-guanidinoprop-1-yl, imidazolylmethyl, orindol-3-yl-ethyl.
 18. The method of claim 6, wherein the peptoidcomprises a backbone modification selected from the group consisting of:N-alkylation, α-ester, thioamide, N-hydroxylation, β-ester, sulfonamide,sulfonamide-N, urea, and urethane.